High-performance liquid chromatography (“HPLC”) instruments are analytical tools for separating, identifying, and quantifying compounds. Traditional HPLC instruments use analytical columns constructed from stainless-steel tubing. Typically, the tubing has an inner bore diameter of 4.7 mm, and its length ranges from about 5 cm to about 25 cm.
In addition, the analytical column of an HPLC instrument typically has a fritted end fitting attached to a piece of tubing. Particles, typically silica-based, functionalized with a variety of functional moieties, pack the tube.
To achieve optimal separation efficiency, using the completed column, an appropriate flow rate of a mobile phase is important. For a 4.7 mm diameter column packed with 5 μm diameter particles, a desirable flow rate is typically between about 1 mL/min and about 2 mL/min. Minimizing the presence of unswept dead volume in the plumbing of the HPLC instrument is desirable for maintaining separation efficiency.
In an HPLC instrument, an injector is typically used to inject a sample into a flowing mobile phase as a discrete fluidic plug. Dispersion of a plug band as it travels to and/or from the column reduces the ultimate efficiency of the chromatographic system. For example, in a chromatographic system using 4.7 mm column tubing and a mobile phase flowing at 1-2 mL/min, tubing having an outer diameter of 1/16 inch (1.6 mm) and an inner diameter of about 0.010 inch (0.25 mm) is typically used to plumb connections between the various HPLC components (e.g. pump, injector, column, and detector). For these flow rates and tubing dimensions, it is relatively easy to machine port details to tolerances that will ensure minimal band broadening at tubing interfaces.
A desire to reduce mobile-phase solvent consumption, in part, has motivated a trend towards reducing column inner diameter. Thus, several scales of chromatography are now commonly practiced; these are typically defined as shown in Table 1 (where ID is inner diameter.)
TABLE 1HPLC scaleColumn IDTypical flow rangeAnalytical4.7mm1s mL/minMicrobore1-2mm100s μL/minCapillary300-500μm10s μL/minNano50-150μm100s nL/min
Microbore HPLC has often been practiced with equipment similar to that used for analytical scale HPLC, with minor modifications. Aside from requiring the exercise of a small degree of additional care in making fittings, microbore HPLC typically requires an operating skill level similar to that of analytical scale HPLC.
In contrast, capillary and nano-scale HPLC require relatively significant changes in HPLC components relative to analytical-scale HPLC. Generation of stable mobile-phase flows of less than about 50 μL/min is relatively difficult using standard open-loop reciprocating HPLC pumps, such as those commonly found in analytical and microbore HPLC systems.
For capillary-scale chromatography, stainless-steel tubing is usable for component interconnections; however, the inner diameter must typically be less than 0.005 inch (less than about 125 μm). Care is generally required in the manufacture of fitting terminations to avoid creation of even minute amounts of dead volume.
For nano-scale chromatography, tubing having inner diameters of about 25-50 μm is typically required to interconnect components of an instrument (e.g. to connect a pump to a separation column). Because stainless-steel tubing is typically unavailable in these dimensions, polyimide-coated fused-silica tubing is typically used. Although fused-silica tubing has excellent dimensional tolerances and very clean, non-reactive interior walls, it is fragile and can be difficult to work with. In addition, interconnection ports should be machined to exacting tolerances to prevent even nanoliters of unswept dead volume.
While the primary motivation to replace analytical-scale HPLC with microbore-scale HPLC may be the desire for reduced solvent consumption, moving to capillary-scale and nano-scale chromatography can support improved detection sensitivity for mass spectrometers, in addition to further reducing solvent consumption, when, for example, flows of less than about 10 μL/min are used. Moreover, capillary-scale or nano-scale systems are often the only options for the sensitive detection typically required for applications involving small amounts of available sample (e.g. neonatal blood screening).
Despite the advantages of capillary-scale and nano-scale chromatography, HPLC users tend to employ microbore-scale and analytical-scale chromatography systems. As described above, these systems typically provide good reliability and relative ease-of-use. In contrast, maintenance of good chromatographic efficiency while operating a capillary-scale or nano-scale chromatographic system requires significant care when plumbing the system (e.g., using tubing to connect pump, injector, column, and detector).
In practice, an operator switching from an analytical or microbore-scale system to a capillary or nano-scale system at times finds that better separation efficiency was achieved with the higher-flow rate (i.e. the analytical or microbore-scale) system. This typically occurs due to insufficiency in the operator's knowledge or experience required to achieve low band-spreading tubing interconnections. Moreover, use of smaller inner-diameter tubing at times can lead to frequent plugging of tubing.
Due the relative difficulty typically encountered with capillary-scale HPLC systems and, even more so, with nano-scale HPLC systems, such systems have primarily been used only when necessary, such as for small sample sizes, and when a relatively skilled operator is available. Thus, analytical laboratories tend to possess more analytical-scale and microbore-scale systems than capillary-scale and nano-scale systems, and do not realize the full benefits available from capillary-scale and nano-scale HPLC.
Separation techniques, such as HPLC, are often utilized in combination with one or more additional analysis techniques, to provide multidimensional information about a sample. For example, mass spectrometry (“MS”) can provide molecular weight and structural information. One problem in combining disparate techniques is provision of sample interfaces.
For example, the combination of LC and MS typically requires transport and ionization of a sample eluent produced by LC, for analysis by MS. Soft ionization techniques, such as field desorption, thermospray and electrospray, are beneficial for production of intact molecular ions that originate from high molecular weight molecules such as proteins and peptides. The precise biological application will often determine a preferred soft-ionization technique.
High-performance liquid chromatography (“HPLC”) instruments are known comprising an installation chamber for receiving a microfluidic cartridge having an electrospray emitter. The microfluidic cartridge houses a substantially rigid ceramic-based multilayer microfluidic substrate (also referred to herein as a “ceramic tile”). Further details of the known ceramic tile arrangement are disclosed in, for example, US 2009/0321356 (Waters Corporation) which is incorporated herein by reference.
For protein samples, the ceramic may comprise a High-Temperature Co-fired Ceramic (“HTCC”) which provides suitably low levels of loss of sample due to attachment of sample to walls of conduits in the substrate. Formed in the layers of the substrate is a channel that operates as a separation column.
Apertures in the side of the substrate provide openings into the channel through which fluid may be introduced into the separation column formed within the ceramic tile. Fluid passes through the apertures under high pressure and flows toward the electrospray emitter coupled at the egress end of the channel. Holes in the side of the microfluidic cartridge provide fluidic inlet ports for delivering high pressure fluid to the substrate.
A problem with the known ceramic-based microfluidic substrate is that it requires a relatively high capillary voltage to be applied to the electrospray emitter for optimum performance. A high capillary voltage increases the likelihood of electrical discharge which may damage and decrease the lifetime of hardware components such as the emitter tip.
WO 00/52455 (Schultz) discloses a droplet/electrospray device and a nano-scale liquid chromatography-electrospray system.
The electrospray device disclosed in WO 00/52455 (Schultz) is microchip-based having a nozzle etched from a surface of a monolithic silicon substrate. FIG. 3E, for example, shows the electrospray device in use wherein an extracting electrode may be held at a voltage Vextract. In an arrangement, the ion-sampling orifice of an API mass spectrometer may function as the extracting electrode. These conventional electrospray devices do not include an integrated liquid chromatography device and do not provide an interface for combined LC and MS analysis.
In this conventional arrangement the extracting electrode is not a distinct counter electrode arranged downstream of the spray emitted from the capillary tip and upstream of an atmospheric pressure interface of a mass spectrometer. In the conventional arrangement, therefore, the ion-sampling orifice of the mass spectrometer functions as an electrode. The conventional arrangement suffers from the problem of a dispersive electric field at the tip of the electrospray emitter and does not help to reduce capillary voltages. Furthermore, the various components of the ion-sampling orifice of the mass spectrometer function as an electrode which is remote from the tip. As a result, a dispersive field is formed which results in ion beam spreading. Furthermore, the strongly divergent electric field lines may terminate on the inner surface of the ion-sampling orifice cone and ions entering the orifice can thus be lost to the inner walls of the cone. As a result, the ion beam has reduced penetration into the first vacuum region of a mass spectrometer and the sensitivity of the mass spectrometer is reduced.
It is apparent, therefore, that the arrangement disclosed in WO 00/52455 (Schultz) is problematic.
FIG. 6C of WO 00/52455 (Schultz) discloses an arrangement wherein a nano-scale liquid chromatography device may be integrated with an electrospray device. The device includes electrodes that are formed on a recessed annular region and on an ejection surface. In this arrangement the various components of the ion-sampling orifice of the mass spectrometer function as an electrode and as a result the electric field at the tip of the electrospray emitter is dispersive which is problematic.
US 2005/0092855 (Li) discloses an arrangement wherein an electrospray apparatus is provided with an auxiliary electrode.
WO 01/91158 (Chen) discloses an arrangement wherein an ion lens is used to focus ions to the inlet of a downstream mass spectrometer.
US 2009/045333 (Chiarot) discloses an electrospray emitter having an electrode and method of using the same.
US 2004/036019 (Goodley) discloses a source of ions for an analyser which includes an electrode.
It is desired to provide an improved interface for a mass spectrometer.